1. Field of the Invention
The present invention relates to apparatus and methods for separating and detecting enzyme substrates using separation gels. For example, the apparatus and methods can be used to separate and detect kinase substrates for further analysis. The apparatus and methods can also be used to detect enzyme inhibitors, such as kinase inhibitors.
2. Background of the Invention
Protein phosphorylation regulates nearly all cellular processes. The enzymes that catalyze the addition of phosphate groups to proteins are termed protein kinases. Deregulated kinase activity has been linked to the pathophysiology of major diseases, including cancer, diabetes, and Alzheimer's disease (Cohen P., Eur J Biochem 268:5001-5010 (2001)). In recent years, kinases have become the focus of intense drug development efforts within the pharmaceutical industry. Currently, kinases are second only to G-protein coupled receptors as targets for development of therapeutics.
A major focus within the pharmaceutical industry is the identification of kinase inhibitors, as well as kinase substrates, and in general, enzyme substrates and inhibitors. The interest in identification of enzyme inhibitors has been fueled by the clinical success of several compounds with kinase inhibitory activity. A prominent example is imatinib, an ABL kinase inhibitor that is extremely effective in treating certain forms of leukemia (Ren R., Nat. Rev. Cancer 5:172-183 (2005)) and gastrointestinal tumors (Blay J Y, et al., Bull Cancer 92:E13-18 (2005)). However, wholesale inhibition of activity of certain kinases may lead to unwanted side effects. As a result, it may be desirable in many cases to block the ability of a kinase to phosphorylate a specific substrate or class of substrates. For example, in Alzheimer's disease, a kinase called GSK3 hyper-phosphorylates the protein Tau, which is a major component of the characteristic neurofibrillary tangles of Alzheimer's disease (Drewes G., Trends Biochem. Sci. 29:548-555 (2004). A major impediment to the development of substrate-specific inhibitors is that for most enzymes such as kinases, the spectrum of substrates that can be phosphorylated is not known. There is currently therefore a need for assays that will facilitate the identification of substrates for enzymes, and specifically kinases. The present invention fulfills these, and other, needs by providing apparatus and methods for separating, detecting and analyzing enzyme substrates, as well as methods for separating, detecting and analyzing inhibitors of enzyme activity.